Canine distemper of domestic dogs is caused by canine distemper virus (CDV), a member of the morbilliviruses. It has been a highly contagious disease of great veterinary importance for centuries, but for the last several decades it has been controlled satisfactorily by modified live vaccines. In the 1990s, however, it was described that CDV strains genetically different from vaccine strains may have caused the disease in vaccinated dogs. The highest antigenic variation is found in the H protein. Therefore, in the present study, hemagglutinin (H) genes obtained from current vaccines and field isolates and amplified directly from clinical specimens were genetically analyzed by restriction fragment length polymorphism assay and sequencing. Phylogenetic analysis of the H-gene amino acid sequences revealed that at least two CDV genotypes are circulating among dogs in Japan; one is a genotype to which almost all Japanese CDV isolates belong and the other has not been previously described. Both are separate and independent from the other lineages or genotypes of vaccine strains, as well as European and U.S. CDV isolates. The results suggest that CDV has also evolved in Japan, and further studies will be needed for an evaluation and possible improvement of the efficacies of current CDV vaccines.

ABSTRACT. Ten strains, eight field and two reference laboratory strains, of canine coronavirus (CCV) were comparatively examined with respect to antigenic relationships and pathogenic potential in dogs. With monoclonal antibodies and hyperimmune antisera to feline coronavirus and CCV, respectively, varying degrees of antigenic diversities were found among the strains by neutralization and immunofluorescence assays, but it was felt that they belong to one serotype. Specific-pathogen-free puppies experimentally inoculated with some CCV strains manifested clinical symptoms, but there was a difference in their virulence. In order to elucidate the prevalence of CCV infections in dogs in Japan, we tested for neutralizing antibodies to CCV in 467 field dogs, and found a prevalence of 44.1%. Moreover, by using nested reverse transcriptase-polymerase chain reaction on rectal swabs of 100 diarrheic dogs recently presented in veterinary clinics, evidence of CCV in 16% of these specimens was found. The results suggested that CCV infection is more widespread than expected in dogs, and that CCV is a significant etiologic factor in canine diarrhea also in Japan.-KEY WORDS: canine, canine coronavirus, diarrhea, enteritis.J. Vet. Med. Sci. 61 (7): [731][732][733][734][735][736] 1999 we examined eight strains newly isolated from diarrheic dogs, and two American strains as reference with respect to their antigenic and pathogenic characteristics. For serological epidemiology of CCV infection, neutralizing antibodies to CCV in dog serum samples recently collected were determined. In addition, we applied nested reverse transcriptase-polymerase chain reaction (RT-nPCR) for detection of CCV in the recent field diarrheic dog feces. MATERIALS AND METHODS Virus isolation and reference CCVs:Isolation of CCV was tried for feces of diarrheic dogs collected from various parts of Japan during the years 1990 to 1997. A rectal swab was placed in 1 ml of Eagle's minimal essential medium (Eagle's MEM), the extract was clarified by centrifugation at 8,500 g for 10 min, and supernatant was inoculated onto the felis cutus whole fetus-4 (fcwf-4) cell monolayer in a culture dish. After 1hr of adsorption, the inoculum was removed and the conditioned Eagle's MEM containing 5% fetal calf serum, 10% tryptose phosphate broth and antibiotics (penicillin 200 U, streptomycin 200 µg and kanamycin 20 µg/ml) was added. Then they were incubated at 37°C in a humidified chamber containing 5% CO2. When a cytopathic effect (CPE) was observed, an isolate was identified by an indirect immunofluorescence assay (IFA) with specific monoclonal antibodies (MAb) to FCoV, as described previously [13].Reference CCV strains 1-71 and TN449 were obtained from Tokyo University, Tokyo and Fort Dodge Laboratories, Iowa, respectively.MAbs and hyperimmune antisera: A MAb reagent, which is a mixture of MAbs against each nucleocapsid, integral membrane and spike protein of FCoV, was used for

No abstract

ABSTRACT. A canine calicivirus (CaCV) isolated in Japan, designated as CaCV No. 48 strain, was propagated in MDCK cells and purified by CsCl equilibrium gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified samples revealed the presence of only one major species of viral protein of about 60 kilodaltons after Coomassie staining. The same band, presumably that of the capsid protein, was detected by western blotting using a mouse hyperimmune serum. This capsid protein was synthesized in MDCK cells as early as 2 hr post-inoculation. Experimental infection of dogs resulted in the production of anti-CaCV antibodies which were detected by microneutralization test and western blotting. Likewise, serosurvey revealed not only the presence of neutralizing antibodies but also reactivity of the field sera against the capsid protein of the purified virus. These results indicate that the capsid protein of CaCV No. 48 strain is immunogenic and could be detected by antibodies in western blotting. -KEY WORDS: canine calicivirus, capsid protein, western blotting.

ABSTRACT. A monoclonal antibody (MAb) reactive with 36 field isolates and 2 laboratory strains of feline calicivirus (FCV) was produced by immunizing mice with the mixture of FCVs. The MAb (4D7) reacted with FCVs in an enzyme-linked immunosorbent assay (ELISA), but had no neutralizing activity against the F4 strain of FCV. MAb 8G1, previously produced against the FCV F4 strain, also reacted in ELISA with all FCVs used in the present study. However, the epitopes recognized by 4D7 and 8G1 were different. Using these two MAbs and a polyclonal rabbit antibody, we attempted to develop a sandwich ELISA for detection of FCV antigen. The combination of 4D7 and the polyclonal rabbit IgG was most sensitive. Using this system, all the field isolates of FCV cultured in vitro were detected. However, among the 36 swab samples, from which FCV was isolated, 4 were negative.-KEY WORDS: feline calicivirus, monoclonal antibody, sandwich ELISA.J. Vet. Med. Sci. 60(2): 155-160, 1998 Serial passages of 9 field isolates, F4 and F9 strains were carried out and the high-passaged viruses, passage number of 100, were also used.The 90-8A strain of FHV1, isolated in 1990 from a cat with respiratory disease, was also used in the present study.Preparation of virus antigen: Virus antigen for immunization and ELISA was prepared from culture fluid of CRFK cells inoculated with each strain of FCV. The culture fluid of FCV was collected and centrifuged at 1,000G for 10 min. For precipitation of the virus, 22.8 g of ammonium sulfate was added to 100 ml of the culture fluid. After centrifugation at 10,000G for 20 min, the virus was suspended in Dulbecco's phosphate buffered saline (PBS) and dialyzed overnight against PBS at 4°C. Following centrifugation at 700G, the protein concentration was measured by using the Bio Rad protein assay (Bio Rad Laboratories, Inc., CA, U.S.A.). For immunization, four types of antigen were prepared. Antigen A was a mixture of all strains of FCV. Antigen B and antigen C were each a mixture of 13 strains of FCV field isolates. Antigen D was a mixture of 10 field isolates, together with the F4 and F9 strains of FCV.Anti-FCV monoclonal antibodies: Six week-old BALB/ cCrSlc mice were immunized with antigen A intraperitoneally followed by a second immunization with antigen A subcutaneously 1 week later. The mice were then immunized subcutaneously with antigen B, C and D at 5 day intervals. The last immunization was performed subcutaneously using antigen A. Three days after the last immunization, the mice were killed and the spleen cells fused with the myeloma cells, P3X63Ag8.653 using polyethylene glycol 1,500 (Boehringer Mannheim GmbH, Germany). Hybridomas were selected using HAT medium Feline calicivirus (FCV) is one of the major pathogens for cat upper respiratory diseases. Although vaccination for FCV has been available for almost twenty years, a high proportion of vaccinated cats present with clinical signs of FCV infection [1]. A number of different strains of FCV have been isolated and the serological relationships...